The highly cytotoxic protein ExoU (74 kDa) from the opportunistic bacterial pathogen Pseudomonas aeruginosa is a major contributor to severe and acute infections. ExoU has been shown to be important for virulence in animal models of pneumonia, and expression of ExoU is closely linked with severe disease and mortality in patients. ExoU is delivered through the bacterial type III secretion system directly into the cytosol of host cells, where it exerts its destructive action. Our long-term objectives are to uncover the mechanism of action of type III secretion effectors, in order to devise means to combat infections caused by type III secretion system-utilizing bacterial pathogens. Recent evidence indicates that the swift and profound cytotoxicity caused by ExoU is due to its action as a phospholipase. However, ExoU is found to be inactive prior to entry into eukaryotic cells, and instead requires a eukaryotic host cell factor for activation as a membrane-lytic, cytotoxic phospholipase. The ExoU-activating host cell factor appears to be conserved from yeast to humans, but to be absent in bacteria. Furthermore, this activation mechanism appears generalizable to the ExoU-related protein RP534 expressed by the bacterial pathogen Rickettsia prowazekii, the cause of epidemic typhus, and may be generalizable to ExoU-related proteins of other bacterial pathogens, such as Legionella pneumophila. This R21 proposal is aimed at identifying the host cell factor responsible for activation of ExoU as a cytotoxic phospholipase. If successful, these studies will be the basis for a longer-term, more extensive project focused on mechanistic principles underlying ExoU activation and possible routes to inhibition of this cytotoxin and related proteins from bacterial pathogens. Our specific aims are to (1) biochemically identify and validate the host cell factor responsible for ExoU activation, and to (2) use yeast genetics to identify and characterize host genes involved in ExoU activation and cytotoxicity. The proposed combination of biochemical and genetic approaches provides a powerful means for understanding steps required for activation of ExoU. The work described here will have potential impact in treating acute infections caused by the opportunistic bacterial pathogen Pseudomonas aeruginosa, and may also have impact on treatment of other bacterial infections. [unreadable] [unreadable] [unreadable] [unreadable]